A fundamental question in immunology is how the innate immune system detects pathogenic microbes. Host health depends critically upon the ability of the innate immune system to respond rapidly and selectively to pathogens, while avoiding inappropriate responses to self or harmless commensal microbes. Our goal is to describe how this rapid, sensitive, and accurate detection of pathogens is achieved at a molecular level. We have been particularly interested in a family of cytosolic pathogen detectors called inflammasomes. Inflammasomes comprise a family of multiprotein complexes that assemble in the cytosol in response to noxious and infectious stimuli. Inflammasomes initiate innate immune responses by activating the Caspase-1 protease. In general, it is poorly understood how inflammasomes recognize pathogens and assemble to activate Caspase-1. To address these questions, our studies have focused on a sub-family of inflammasomes called the Naip/Nlrc4 inflammasomes. Naip proteins (of which there are four in C57BL/6 mice) and Nlrc4 are members of a broader superfamily of proteins called NLRs (nucleotide- binding domain, leucine rich repeat-containing proteins). In the past funding period, we found that Naip5 mediates specific cytosolic detection of bacterial flagellin, whereas Naip2 mediates specific cytosolic detection of the inner rod protein from diverse bacterial type III secretion systems. We have also developed novel biochemical assays to dissect ligand binding and inflammasome oligomerization, and have used these novel assays to show that a key biochemical function of the Naips is to recognize ligands and induce downstream oligomerization of Nlrc4. In this renewal application, we describe three aims that will dissect ligand binding, oligomerization, and the downstream in vivo effector functions of the Naip/Nlrc4 inflammasomes. Aim 1 seeks to determine how specific bacterial ligands are recognized by Naip proteins. This will be significant because it will represent the first analysis of ligand binding by any mammalian NLR protein. Aim 2 seeks to dissect the biochemical and cellular mechanisms of inflammasome assembly, a critical but poorly understood process. Lastly, Aim 3 will investigate how inflammasomes initiate effector responses in vivo. In particular, we seek to establish a novel link between inflammasome activation and eicosanoid lipid mediator production in vivo. Eicosanoids are known to be critical mediators of inflammation, but their production has not previously been linked to inflammasomes. We show in vivo that inflammasome activation results in eicosanoid production and a severe vascular leakage syndrome that can be rapidly fatal. Taken together, our studies of the Naip/Nlrc4 inflammasomes will provide novel insights into ligand recognition, assembly and in vivo function of this critical family of cytosolic immune detectors.